Ethylnitrosourea (ENU) is a powerful transplacental carcinogen. A single dose to a pregnant rat produces neural tumors in adult offspring many months later. The long latency period offers an opportunity to look for early oncogenic changes in target neural tissues, applying a combination of in vivo and in vitro techniques. We have demonstrated that tumorigenic doses of ENU produce chromosome aberrations in bone marrow and fetal tissues of treated animals as well as in cultured cells in vitro, suggesting that a mutation could be an indication, and possibly an explanation, for the initial oncogenic effect. Rat neural tumors, produced by ENU have been grown in cell culture. In cell lines from the gliomas and Schwannomas we have compared chromosome constitution, growth characteristics in culture, histological findings of the tumor and tumorigenicity of the cells. In 12 tumor lines excess chromosome 4 was present as a common chromosome finding, suggesting that extra chromosome 4 can be useful as a marker for ENU neural tumors. Our main objective is to study early stages of oncogenesis in neural target tissues. Brain and trigeminal nerve ganglia are removed from rats treated transplacentally with ENU. At various stages postnatally neural tissues are initiated in cell culture and monitored for oncogenic transformation, cytogenetic changes and tumorigenicity. We will attempt to determine whether excess chromosome 4 is related to ENU as a specific etiologic agent, by studying the chromosome constitution of non-neural ENU tumors, or whether the chromosome is associated with a particular tumor cell type, by studying rat brain tumors induced by other agents. The role of chromosome 4 in initiation or progression of the tumors will also be investigated by examining direct preparations of tumors with special emphasis on a search for competing cell lines and subclones.